We study the mechanism and regulation of protein synthesis in eukaryotic cells focusing on regulation by GTP-binding (G) proteins and protein phosphorylation. The first step of protein synthesis is binding the initiator Met-tRNA to the small ribosomal subunit by the factor eIF2. The eIF2 is a GTP-binding protein and during the course of translation initiation the GTP is hydrolyzed to GDP. The eIF2 is released from the ribosome in complex with GDP and requires the guanine-nucleotide exchange factor eIF2B to convert eIF2-GDP to eIF2-GTP. This exchange reaction is regulated by a family of kinases that specifically phosphorylate the alpha subunit of eIF2 on serine at residue 51, and thereby covert eIF2 into an inhibitor of eIF2B. Among the family of eIF2alpha kinases are GCN2 (activated under conditions of amino acid starvation), PKR (activated by double-stranded RNA and downregulates protein synthesis in virally infected cells), and PERK (activated under conditions of ER stress). Our previous molecular genetic studies of PKR were useful for uncovering how dimerization of PKR serves to regulate catalytic activation;therefore we employed a similar strategy to gain insight into how PKR gains access to the Ser51 phosphorylation site in eIF2alpha. In the crystal structure of the PKReIF2alpha complex, the C-terminal lobe of the kinase contacts eIF2alpha on a face remote from Ser51, leaving Ser51 20 angstroms from the kinase active site. PKR mutations, such as PKR-T487A, that cripple the eIF2alpha-binding site impair phosphorylation, and we identified mutations in eIF2alpha that suppress the PKR-T487A mutation, increase Ser51 loop mobility, and restore phosphorylation both in vivo and in vitro. These eIF2alpha mutations either disrupt a hydrophobic network that restricts the position of Ser51 or alter a linkage between the PKR-docking region and the Ser51 loop. Finally, we found that addition of PKR increases the protease sensitivity of the Ser51 loop in eIF2alpha. We propose that restricted mobility of Ser51 in free eIF2alpha prevents promiscuous phosphorylation and the attendant translational regulation by heterologous kinases, whereas binding of PKR to eIF2alpha triggers a signal that induces a conformational change of the phosphorylation site sequences and enables Ser51 to access the PKR active site. In order to subvert the anti-viral defense mediated by PKR viruses produce inhibitors of the kinase. Several members of the poxvirus family express two different types of PKR inhibitors: a pseudosubstrate inhibitor (such as the vaccinia virus K3L protein that resembles the N-terminal third of eIF2alpha) and a double-stranded RNA binding protein called E3L. High-level expression of human PKR inhibited the growth of yeast, and co-expression of the vaccinia virus K3L or E3L protein or the related variola (smallpox) virus C3L or E3L protein, respectively, restored yeast cell growth. We identified PKR mutations that confer resistance to K3L inhibition but do not affect eIF2alpha phosphorylation, demonstrating that subtle changes to the PKR kinase domain can drastically impact pseudosubstrate inhibition while leaving substrate phosphorylation intact. We proposed that these paradoxical effects on pseudosubstrate versus substrate interactions reflect differences between the rigid K3L protein and the plastic nature of eIF2alpha around the Ser51 phosphorylation site. Phylogenetic analyses of the eIF2alpha kinases plus four unrelated protein kinases revealed fast evolution of the PKR kinase domain in vertebrates. Substitution of positively selected (diversified) residues in human PKR with residues found in other species altered the sensitivity to PKR inhibitors from different poxviruses. Our results indicate how an antiviral protein (PKR) evolved to evade viral inhibition while maintaining its primary function (phosphorylation of eIF2alpha). Moreover, our identification of species-specific differences in human versus mouse PKR susceptibility to viral inhibitors has important implications for studying human infections in nonhuman model systems. The GTP-binding protein eIF5B catalyzes ribosomal subunit joining in the final step of translation initiation. Our previous studies revealed that GTP hydrolysis by eIF5B activates a regulatory switch required for eIF5B release from the ribosome following subunit joining. Analysis of rRNA mutations that suppress eIF5B mutants that lack GTP hydrolysis activity identified a functionally important docking between domain II of eIF5B, which is conserved in other translational GTPases, and the body of the small ribosomal subunit. The eIF5B resembles a chalice with the &#945;-helix H12 forming the stem connecting the GTP-binding domain cup to the domain IV base. Helix H12 has been proposed to function as a rigid lever arm governing domain IV movements in response to nucleotide binding and as a molecular ruler fixing the distance between domain IV and the G domain of the factor. To investigate its function, we altered the length and rigidity of helix H12. Whereas helix H12 mutations had minimal impacts on GTP binding and on eIF5B ribosome binding and GTPase activities, shortening the helix impaired the rate of subunit joining in vitro and both shortening the helix and increasing its flexibility impaired the stability of Met-tRNA bound to the 80S product of subunit joining. These data support the notion that helix H12 functions as a ruler connecting the GTPase center of the ribosome to the P site where Met-tRNA is bound and that helix H12 rigidity is required to stabilize Met-tRNA binding. The translation factor eIF5A, the sole protein containing the unusual amino acid hypusine N&#949;-(4-amino-2-hydroxybutyl)lysine, was originally identified based on its ability to stimulate the yield (endpoint) of methionyl-puromycin synthesis, a model assay for first peptide bond synthesis. However, the precise cellular role of eIF5A is unknown. Using molecular genetic and biochemical studies, we recently showed that eIF5A promotes translation elongation, and this activity is dependent on the hypusine modification. As eIF5A is a structural homolog of the bacterial protein EF-P, we propose that eIF5A/EF-P is a universally conserved translation elongation factor.